Netherlands Brain Bank tissue request continued (Sample transfer process / MTA / Transportation / cost per sample / IHC paraffin vs frozen / Molecular analysis / Location-Region-Analysis-Brain Amount table / Tony Wolinsky purchasing flow / NBB blood-cells correction / genetic testing / segregation in the family / pathogen testing / NLRP3 plan / AD co-pathologies / Braak stage classification opener)

Netherlands Brain Bank — tissue request flow

For ad/professional organizations one tissue block is 1 unit. Paraffin embedded sample upon autopsy tissue is fixed for about 30 days in 4% formaldehyde, then the tissue sample is embedded in paraffin. Sample size approximates 1 gram, on request (& depending on committees decision), they may provide the block or they generate FFPE section (10 um as below). Paraffin and frozen tissue blocks: 1 tissue block is equal to 1 unit. CSF is allowed and is supplied as 1 ml aliquots and 8 contains 50 µl with a concentration of 10 ng/ml. DNA would cost €256.50 for the profile.

CSF samples can also be obtained from researchers at the same time of donation, including genetic analysis, as it is included in the informed consent. As we facility, there is no pathologic or histologic screening of the samples, but we handle all material as potentially hazardous. However, if our pathologist would handle any doubts about for example prion disease, HIV infection… the donor would not be accepted for autopsy at the NBB.

[Sample transfer process] https://www.brainbank.nl/brain-tissue/application/

Reviewed by the NBB’s scientific committee. Within 6 weeks upon receipt of the application form, we aim to inform you whether your request can be approved.

• a project number is assigned
• the researcher is informed about the subsequent procedure for obtaining the requested samples and the financial contribution.
• CSF Invoicing & payment FAQ

[MTA] The MTA must be signed by an authorized representative of the university or institute in duration. Our master MTA per research institute (legal entity) covers all legal terms of delivery. Based on this MTA, all researchers in the research institute may apply for tissue samples.

A Material Transfer Statement (MTS), a clear summary of the MTA, is sent to the researcher along with an implementing Letter. This letter of approval specifies the material that will be supplied and the financial contribution.

Upon receipt of the implementing Letter and MTS, both signed by the applicant, the material will be supplied.

Transportation

The transportation method depends on the storage type(s) of the selected tissue. Please note that frozen tissue samples are shipped on dry ice, which means that they are exposed to CO2 despite the fact that the samples are sealed in airbags. Many people who handle samples like ours do not know about the additional toxic CO2 exposure. Therefore, depending on the type of analysis to be performed on the sample, recipients may need to allow the samples to outgas before opening them. NBB will retain a small amount of CO2-free environment after the first opening to allow normalize to a freezer for 96 hours prior to performing the assay. To allow the CO2 to dissipate. If it is not possible to wait for 96 hours, a second solution is to remove the cap and vent the CO2 before thawing the sample. A summary of the article by Murphy et al. can be found here.

cost per sample

	Brain	Plasma
minimum that can be requested	4 frozen blocks (request) 1 ml	2 ml are equal to 1 unit, lasma evenly contain 1 ml, and therefore £22 / 4 unit
cost per sample (£)	£275	£275
	4	22
Total cost (£)	18,700	4,675 / 9,350 / £32,725

IHC paraffin preferred / frozen + Tissue

There is a protocol though Tom (http://www.minerals.dev). Frozen tissue sections are fixed with alcohol following cryopreservation. Each method has distinct advantages and disadvantages. While paraffin embedding is thought to better preserve morphological details, cryopreservation is considered to better preserve everything antigen mostly.

Molecular analysis

Challenging due to extensive formalin-induced cross-linking of proteins. Preferred. For molecular analyses, such as mass spectrometry, quantitative real-time PCR, next-generation sequencing and Western blotting for PTMs, frozen samples are a necessity.

Location / Region / Analysis / Brain Amount

#	Location	Region	Analysis	Brain Amount
1	TSHO	Parkin GT	MBM (Parkin, pS65-Ub, TOM40/20, mtDNA)	100-200 mg total
2	TBOS	Parkin GT	Imaging (MC1)	200 mg & 5 cm × 1 cm × 1 cm
3	TBOS	NLRP3	Imaging (NLRP3)	100 mg & 5 cm × 1 cm × 1 cm
4	TCAL	NLRP3	MBM (NLRP3 and inflammation?)	200 mg

Purchasing flow

1. Decide Recipient’s name and address who will receive the tissues from Tbos (this info is needed for “Appendix B Implementing Letter”) - Chris Leonard, MA, MTL, Takeda Development Center Americas, Inc, 35 Lansdowne Street, Room 35-5052A, Cambridge, MA 02139-4232, [email protected] 617-552-8866
2. Tony Wolinsky creates a purchasing order via US Ariba, using the cost center of NSTM-Shimar 2780002165 1 (Once Tony’s line manager approves it). Purchasing Order number is generated. (assuming this process is the same as Japan Ariba)
3. Tony Wolinsky shares the following information with Jaewon: Tony Wolinsky’s address, Purchasing Order number.
4. Jaewon fills in and sends Appendix B (Implementing Letter) to NBB
5. NBB sends the tissues to the Recipient at Tbos.
6. Once the recipient let NBB know of the receipt of the tissues, NBB sends Tony Wolinsky the invoice.
7. Tony Wolinsky proceeds the payment via US IVF (I am not sure whether Tcal also uses IVF for payment).

We got a correction from NBB that these ‘bloodcell samples’ are a mixture of blood cells (WBC, RBC, platelets) and buffy coat, not pure mononuclear cell samples.

Genetic testing

	content
a deletion-specific PCR test	cDNA research
cDNA research	cDNA research is difficult if it concerns a deletion of the first exon. In addition, I estimate that the chance that there may be cDNA research is no longer the case is about 1/300. We need cDNA. Do you have cDNA preferable from cell lines treated with cyclodemixide? If yes, we need to develop specific cDNA primers, but without the size of the per product should be fine. If you do not have cDNA, we could look into long read sequencing. This is something we are starting with. We have both pacbio and nanopore but this is completely new research and we need to check which protocols are available. This will both take money and time and no guarantees
segregation in the family	This is what we do by default within diagnostics. If the parents are available, they are tested for carrier status. If the parents are not available, any offspring will be tested. If both deletions are on the different alleles, you will show home or 1 of both deletions with each offspring

Below is NBB’s reply on pathogen testing

At our facility there is no pathologic testing/screening of the samples; but we handle all material as potentially hazardous. However, if our pathologist would have any doubts about for example prion disease, HIV infection… the donor would not be accepted for autopsy at the NBB.

(Tay-Jay) My plan is exactly as Shaina suggested, to group us together to discuss

1. likelihood to leverage the original experimental plan to incorporate assessment of the related sample tissue (eg from another sample panel that we can include NLRP3)
2. NLRP3 program to propose other analysis required to support target validation
3. We have current batch of samples can not be used to support NLRP3. Joewon can purchase separate AD samples from NBB through TSHO Ariba. As you can see from one of sample lists (attached), many of the samples are pieces of raw cerebellum. I imagine you can probably use these samples for your NLRP3 studies. We will want the FFPE blocks and CSF for the experiments I mentioned before. In total there are 128 dossied and normal patient samples.

AD co-pathologies Reverse Translation

(Genotype 1 → ApoE3 risk score → Pathogenic Risk Score → Apo, the genotype, and so on)

Braak stage classification (start)

stage	criteria	thal phase	striatum	x	x	ex	on / and / m
Thalamic phase		1	2	3	4	5
B0	no NFTs						2 Probable yes
B1	Braak stage I or II						3 Possible no
B2	Braak stage III or IV						4 No AD no
B3	Braak stage V or VI